The neural crest (NC) plays a critical role in the developmental of the vertebrate head, face and jaws, providing the bulk of the craniofacial skeleton as well as peripheral nervous system and other cranial tissues. Normal craniofacial development depends on proper induction, migration and differentiation of NC cells and derivatives. Deficiencies at any of these steps, whether due to intrinsic defects in NC itself, or in failure of NC cells to interact properly with adjacent tissues, can lead to birth defects: up to a third of all congenital malformations in human neonates are craniofacial in nature and these are primarily due to such NC failures. We have used the freshwater tropical fish Danio rerio (zebrafish) as an experimental model organisms to study NC development. In the past year the lab has focused on the Distal-less (Dlx) family of homeodomain transcription factors. There are 6 Dlx genes in mammals, all of which have orthologues in zebrafish (in addition, the zebrafish genome includes extra copies of Dlx2 and Dlx4). Data from mouse knockout experiments, as well as information from naturally-occurring mutations in human patients, show that the Dlx genes are important in craniofacial development. We are using loss-of-function approaches to investigate Dlx gene function in fish embryos, carried out by gene targeting via CRISPR/Cas methodology. We have identified INDEL mutations in all six primary Dlx genes, with disruption of the additional Dlx2/4 genes in progress. In addition, double mutations in both genes of paired loci, i.e. Dlx1/2, Dlx3/4 and Dlx5/6 have also been isolated. We are in the process of breeding homozygous null embryos by in-crossing. These are in a transgenic background in which early cranial cartilage cells are labelled with GFP, enabling confocal microscopic imaging of live, mutant embryos at all stages. So far, of the single mutant F3 offspring, only Dlx2a disruption exhibits a discernible phenotype. Dlx5/6 double mutants die 8-9 days postfertilization, apparently because they are unable to feed due to jaw defects and possibly also other deficiencies due to Dlx gene function in the brain. We anticipate these studies will reveal some of the earliest events that lead to Dlx-dependent craniofacial dysmorphology, with significant impact on the diagnosis and ultimately on developing therapy for affected human fetal and neonatal patients